How to avoid contamination by keratins ?
It is necessary to take all precautions described below during all manipulation steps of your sample to limit the contamination by keratins that would be detrimental to your results
- avoid to wear whool clothes
- attach long hairs
- wear a clean white coat and take care to correctly cover your forearm
- clean the lab bench used for your experiments with ethanol
- avoid to bend your face over your samples and wear a surgery mask if necessary
- use perfectly clean equipments (equipments for migration, box for coloration, instruments for gel bands excision, tubes free from dust, etc.) : whash with soapy hot water and rince with deionised water
- use solutions and equipments dedicated to mass spectrometry analysis and do not use, particularly, equiments used for western-blot experiments.
Protein extraction is a crucial step for proteomics analysis. If you realise protein extraction by yourself, we can give you some protocols.
Migration on 1D or 2D electrophoresis gels
- Gel type :
The use of precast gels and commercial buffers limit the risks of contamination by keratins. If you prepare your gels by yourself, please follow manufacturer's instructions to avoid contaminations by keratins.
- Quantity of sample :
We recommend to deposit between 20 and 50 µg of total proteins per 1D gel well, Nous recommandons de déposer entre 20 et 50 µg de protéines totales par puits en gel 1D, between 60 and 120 µg for a 2D gel stained with NiAg, between 100 and 600 µg for a 2D gel stained with Coomassie blue.
- Gel staining
Nerver use boxes that have been employed for Western Blot experiments to avoid contaminations by proteins that have been used for membrane saturation (casein-, albumin,…) or plastic boxes to avoid contaminations by polymers !
- Coomassie blue staining :
Use colloidal Coomassie blue because it is compatible with mass spectrometry analysis.
- Silver staining :
Conventional NiAg staining is not compatible with mass spectrometry analysis. Therefore, it is possible to realise a NiAg staining compatible with mass spectrometry by using a specific protocol.
Please, consult us if you wish to perform a Silver staining.
Be carefull : the NiAg staining is very sensitive and can corresponds to a quantity of proteins which is too low to provide protein identification by mass spectrometry. We recommend the use of Coomassie blue staining.
Excision of gel bands/spots
The band excision step is paticularly critical for contaminations by keratins :
- carefully clean the glass plate that will be used for bands excision with ethanol
- wear clean cloves and a surgery mask
- use a scalpel washed with ethanol
If you are interested in a particular gel band, this band will have to be cut as precisely as possible to avoid contamination by proteins present in an adjacent band. Then, the gel band can be cut in small cubes of few mm² and put in an Eppendorf tube.
- If you deliver the sample by yourself : put the gel band at 4°C in an Eppendorf tube filled with deionised water for immediate use or with deionised water/acetic acid 100/0.1 for long-time conservation at -20°C.
- If you send us the samples : put the gel band in an Eppendorf tube filled with deionised water/acetic acid 100/0.1 at room temperature and well protected (by a bubble wrap for example).
In all cases, join the "sample preparation form".